Phase Ia Clinical Evaluation of the Safety and
Immunogenicity of the Plasmodium falciparum Blood-
Stage Antigen AMA1 in ChAd63 and MVA Vaccine
Susanne H. Sheehy1*, Christopher J. A. Duncan1, Sean C. Elias2, Sumi Biswas2, Katharine A. Collins2,
Geraldine A. O’Hara1, Fenella D. Halstead2, Katie J. Ewer2, Tabitha Mahungu3, Alexandra J. Spencer2,
Kazutoyo Miura4, Ian D. Poulton1, Matthew D. J. Dicks2, Nick J. Edwards2, Eleanor Berrie5, Sarah Moyle5,
Stefano Colloca6, Riccardo Cortese6,7, Katherine Gantlett1, Carole A. Long4, Alison M. Lawrie1, Sarah C.
Gilbert2, Tom Doherty3, Alfredo Nicosia6,7, Adrian V. S. Hill2, Simon J. Draper2
1Centre for Clinical Vaccinology and Tropical Medicine, Churchill Hospital, Oxford, United Kingdom, 2The Jenner Institute, University of Oxford, Oxford, United Kingdom,
3University College London Clinical Research Facility, University College Hospital, London, United Kingdom, 4Laboratory of Malaria and Vector Research, National
Institute of Allergy and Infectious Diseases/National Institutes of Health, Rockville, Maryland, United States of America, 5Clinical Biomanufacturing Facility, University of
Oxford, Churchill Hospital, Oxford, United Kingdom, 6Okairo `s AG, Rome, Italy, 7CEINGE, Naples, Italy
Background: Traditionally, vaccine development against the blood-stage of Plasmodium falciparum infection has focused
on recombinant protein-adjuvant formulations in order to induce high-titer growth-inhibitory antibody responses.
However, to date no such vaccine encoding a blood-stage antigen(s) alone has induced significant protective efficacy
against erythrocytic-stage infection in a pre-specified primary endpoint of a Phase IIa/b clinical trial designed to assess
vaccine efficacy. Cell-mediated responses, acting in conjunction with functional antibodies, may be necessary for immunity
against blood-stage P. falciparum. The development of a vaccine that could induce both cell-mediated and humoral
immune responses would enable important proof-of-concept efficacy studies to be undertaken to address this question.
Methodology: We conducted a Phase Ia, non-randomized clinical trial in 16 healthy, malaria-naı ¨ve adults of the chimpanzee
adenovirus 63 (ChAd63) and modified vaccinia virus Ankara (MVA) replication-deficient viral vectored vaccines encoding
two alleles (3D7 and FVO) of the P. falciparum blood-stage malaria antigen; apical membrane antigen 1 (AMA1). ChAd63-
MVA AMA1 administered in a heterologous prime-boost regime was shown to be safe and immunogenic, inducing high-
level T cell responses to both alleles 3D7 (median 2036 SFU/million PBMC) and FVO (median 1539 SFU/million PBMC), with a
mixed CD4+/CD8+phenotype, as well as substantial AMA1-specific serum IgG responses (medians of 49 mg/mL and 41 mg/
mL for 3D7 and FVO AMA1 respectively) that demonstrated growth inhibitory activity in vitro.
Conclusions: ChAd63-MVA is a safe and highly immunogenic delivery platform for both alleles of the AMA1 antigen in
humans which warrants further efficacy testing. ChAd63-MVA is a promising heterologous prime-boost vaccine strategy
that could be applied to numerous other diseases where strong cellular and humoral immune responses are required for
Trial Registration: ClinicalTrials.gov NCT01095055
Citation: Sheehy SH, Duncan CJA, Elias SC, Biswas S, Collins KA, et al. (2012) Phase Ia Clinical Evaluation of the Safety and Immunogenicity of the Plasmodium
falciparum Blood-Stage Antigen AMA1 in ChAd63 and MVA Vaccine Vectors. PLoS ONE 7(2): e31208. doi:10.1371/journal.pone.0031208
Editor: Denise L. Doolan, Queensland Institute of Medical Research, Australia
Received July 27, 2011; Accepted January 4, 2012; Published February 21, 2012
Copyright: ? 2012 Sheehy et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the European Malaria Vaccine Development Association (EMVDA), a European Commission FP6-funded consortium (http://
www.emvda.org/) [LSHP-CT-2007-037506]; the UK National Institute of Health Research through the Oxford Biomedical Research Centre (http://www.oxfordbrc.
org/) [A91301 Adult Vaccine]; and the Wellcome Trust (http://www.wellcome.ac.uk/) [084113/Z/07/Z]. The GIA work was supported by the PATH Malaria Vaccine
Initiative (MVI; http://www.malariavaccine.org/) and the Intramural Program of the National Institutes of Health, National Institute of Allergy and Infectious
Diseases (http://www.niaid.nih.gov/Pages/default.aspx). CJAD holds a Wellcome Trust Research Training Fellowship [RTEI0]; SCG, AVSH and SJD are Jenner
Investigators; AVSH was supported by a Wellcome Trust Principal Research Fellowship [45488/Z/05]; and SJD is a UK Medical Research Council Career
Development Fellow [G1000527]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: AJS, MDJD, SCG, AVSH and SJD are named inventors on US 12/595 574 and UK PCT/GB2008/01262, US 12/595 351 and UK PCT/GB2008/
01271 novel adenovirus patent applications covering malaria vectored vaccines and immunization regimes. This does not alter our adherence to all the PLoS ONE
policies on sharing data and materials. Authors from Okairo `s are employees of and/or shareholders in Okairo `s, which is developing vectored vaccines for malaria
and other diseases. This does not alter our adherence to all the PLoS ONE policies on sharing data and materials.
* E-mail: firstname.lastname@example.org
PLoS ONE | www.plosone.org1 February 2012 | Volume 7 | Issue 2 | e31208
An effective vaccine against the blood-stage of Plasmodium
falciparum infection could significantly contribute to any future
strategy for reducing malaria morbidity and mortality, limiting
transmission and aiding disease eradication . Although anti-
disease and anti-parasitic immunity is naturally acquired against
blood-stage infection following repeated exposure , replicating
such immunity by vaccination has proved extremely difficult .
There have been recent reports of efficacy observed in
retrospective/post-hoc analyses from Phase Ib safety and immu-
nogenicity trials of a blood-stage vaccine  or one with a blood-
stage component . More encouragingly, significant strain-
specific efficacy was also recently reported in a pre-specified
secondary analysis of a Phase IIb trial of a mono-valent 3D7 strain
apical membrane antigen 1 (AMA1) protein vaccine . Of note,
this vaccine also showed an encouraging signal in a prior Phase IIa
controlled human malaria infection study . However, despite
these extensive efforts to date, no candidate blood-stage vaccine
has been developed that has demonstrated statistically significant
efficacy with regard to clinical outcome in a pre-specified primary
endpoint analysis in a Phase IIa/b clinical trial designed to assess
vaccine efficacy [3,8]. The majority of such blood-stage vaccine
candidates have traditionally focused on recombinant protein-in-
adjuvant formulations with the aim of inducing growth inhibitory
antibody responses against merozoite antigens involved in the
erythrocyte invasion process . However, increasing evidence
suggests that T cells can also play an important contributory role
in the mediation of immunity against blood-stage antigens [9,10].
The mechanisms by which T cells could contribute to protective
outcome in vivo in humans remain less well defined, particularly
given the lack of MHC molecules necessary for antigen
presentation on red blood cells (RBCs). One suggestion is that
macrophages in the spleen, activated by cytokines from T helper 1
(Th1)-type CD4+cells specific for blood-stage antigens, may
enhance phagocytic clearance of infected RBCs [11,12]. Another
proposal is that CD4+Th1 cells may bias the induction of
cytophilic antibody subclasses from B cells that can in turn mediate
anti-parasitic neutrophil respiratory burst activity (ADRB)  or
antibody-dependent cellular inhibition (ADCI)  via mono-
cytes. Alternatively, CD8+T cell responses against blood-stage
antigens could target late liver-stage parasite forms which also
express classical ‘blood-stage’ antigens [15,16,17]. An effective
blood-stage vaccine may therefore be required to induce strong
cellular immunity that can act in concert with anti-parasite
Recently, viral vectored vaccines encoding blood-stage antigens
have been developed which can induce potent humoral and
cellular immune responses in animal models . Heterologous
prime-boost immunization with human or simian adenovirus
followed by the orthopoxvirus modified vaccinia virus Ankara
(MVA) expressing the blood-stage antigen AMA1 is highly
immunogenic for both antibodies and T cells in mice, rabbits
 and rhesus macaques . Although a long-standing subunit
vaccine candidate antigen that is susceptible to strain-specific
antibodies , AMA1 exhibits extreme polymorphism 
which has proved a significant obstacle in the development of a
broadly neutralizing antibody-inducing vaccine for use in endemic
populations . In the study reported here, the simian adenovirus
and MVA vectors were designed to express an optimized
transgene encoding two divergent alleles (3D7 and FVO) of
AMA1 [19,20]. These vectors, when used in heterologous prime-
boost regimes in animal models, induced antibodies that mediate
in vitro growth inhibition against both 3D7 and FVO strain P.
falciparum parasites [19,20]. Moreover, similar vaccines, encoding
the orthologous gene, can confer blood-stage efficacy in the P.
chabaudi rodent malaria model, which is dependent on vaccine-
induced antibodies as well as AMA1-specific CD4+T cells (Biswas
et al., submitted). T cell epitopes within AMA1 that elicit
proliferative T cell responses have also been described in
naturally-exposed individuals from Kenya [23,24]. A recent Phase
Ia study of a candidate human adenovirus serotype 5 (AdHu5)
vaccine expressing P. falciparum AMA1 (3D7 strain allele)  was
also shown to be immunogenic for AMA1-specific CD4+and
CD8+T cells in malaria-naı ¨ve adults [26,27]. However, concerns
regarding pre-existing anti-vector immunity to human adenoviral
serotypes [28,29], and the inclusion of just one allele (3D7) in this
vaccine formulation is likely to limit the widespread utility of this
The replication-deficient chimpanzee adenovirus 63 (ChAd63)
has been shown to be a safe, versatile and exceptionally
immunogenic vector when administered in a heterologous
prime-boost regimen with the attenuated orthopoxvirus MVA in
two Phase Ia clinical trials in healthy malaria-naı ¨ve adults in the
UK; one using vectors encoding the liver-stage antigen thrombos-
pondin related adhesion protein fused to a multi-epitope string
(ME-TRAP) (O’Hara et al., J Infect Dis 2011 in press), and the
other using the same vectors encoding the blood-stage antigen
merozoite surface protein 1 (MSP1) . Here we present the
safety and immunogenicity results of the third ChAd63 vector to
be trialled alone and in a prime-boost regimen with MVA. This
Phase Ia trial utilized the ChAd63 and MVA vectors expressing an
optimized bi-valent AMA1 insert designed to address antigen
polymorphism, administered in a heterologous prime boost
regimen to healthy malaria-naı ¨ve adults.
The objective of the study was to assess the reactogenicity and
immunogenicity of ChAd63 AMA1 administered alone and with
MVA AMA1 in healthy malaria-naı ¨ve adults.
The study was conducted at the Centre for Clinical Vaccinology
and Tropical Medicine, University of Oxford, Oxford, UK and
the University College London Clinical Research Facility,
London, UK. Healthy, malaria-naı ¨ve males and non-pregnant
females aged 18–50 were invited to participate in the study. There
was no selection of volunteers on the basis of pre-existing
neutralizing antibodies (NAb) to the ChAd63 vector prior to
enrolment, however NAb titers were subsequently assayed (see
Supplementary Information S1 for the full list of inclusion and
This was a Phase Ia open-label, non-randomized blood-stage
malaria vaccine trial. The clinical trial protocol and supporting
CONSORT checklist are available as Supplementary Informa-
tion; see Protocol S1, Checklist S1 and Supplementary Informa-
tion S1. Allocation to study groups (Figure 1) occurred at screening
based on volunteer preference, as previously described . Eight
volunteers were vaccinated with 56109viral particles (vp) ChAd63
AMA1 (diluted in 0.9% NaCl and administered in 300 mL) (groups
1A & 1B). Four of these volunteers were subsequently vaccinated
in the opposite arm 56 days later with 56108plaque forming units
(pfu) MVA AMA1 undiluted and administered in 200 mL (group
1B). Another eight volunteers were vaccinated with 561010vp
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ChAd63 AMA1 undiluted and administered in 300 mL (group 2A
& 2B) and four of these were subsequently vaccinated 56 days later
in the opposite arm with either 2.56108pfu MVA AMA1
undiluted and administered in 100 mL (Group 2B(i), n=1) or
1.256108pfu MVA AMA1 undiluted and administered in 50 mL
(Group 2B(ii), n=3). Note the dose of MVA AMA1 was reduced
following greater than expected reactogenicity when using
56108pfu in Group 1B. All vaccinations were administered
intramuscularly (IM) into the deltoid.
Volunteers attended clinical follow-up at days 2, 14, 28, 56 and
90 following ChAd63 AMA1 immunization in groups 1A and 2A
and at days 2, 14, 28, 56, 58, 63, 84 and 140 following ChAd63-
MVA AMA1 immunization in groups 1B and 2B. Safety
assessments, including blood sampling for safety and immunology
analysis at these visits were conducted as previously described .
A time window ranging between 1 and 14 days was allowed for
vaccination and follow-up visits. Throughout the paper, study day
refers to the nominal time point for a group and not the actual day
The first volunteer to receive each dose of ChAd63 AMA1 was
vaccinated in isolation. Following a review of reactogenicity in
these individuals 48 hours post vaccination, the remaining
volunteers in each group were enrolled. There was a 2 week
interval prior to dose escalation of ChAd63 AMA1, during which
time a scheduled review of safety data was conducted by the
independent Local Safety Committee.
This was an observational and descriptive study to assess the
safety and immunogenicity of ChAd63 AMA1 and MVA AMA1.
The sample size (n=16) was chosen to allow determination of the
magnitude of the primary outcome measures, especially of serious
and severe adverse events (AEs), rather than assessment of
statistically significant differences between groups.
Ethical & Regulatory Approval
The clinical trial protocol and associated documents were
approved by the UK Gene Therapy Advisory Committee (GTAC
142). Clinical Trial Authorisation was granted by the United
Kingdom Medicines and Healthcare Products Regulatory Agency
(MHRA. Ref: 24821/85158/19/720). Vaccine use was authorized
by the Genetically Modified Organisms Safety Committee
(GMSC) of the Oxford Radcliffe Hospitals NHS Trust, UK
(Reference number GM 462.09.42). All participants gave written
informed consent prior to any study procedure being undertaken.
The study was conducted according to the principles of the
Declaration of Helsinki (2008) and the International Conference
on Harmonization (ICH) Good Clinical Practice (GCP) guidelines.
The Local Safety Committee provided safety oversight and GCP
compliance was independently monitored by an external organi-
zation (Appledown Clinical Research Ltd, Great Missenden, UK).
ChAd63 AMA1 and MVA AMA1 Vaccines
Generation of the recombinant vectors has been previously
described [19,20]. They were manufactured under Good Manu-
facturing Practice conditions by the Clinical Biomanufacturing
Facility, University of Oxford (ChAd63 AMA1) and IDT
Biologika, Rosslau, Germany (MVA AMA1). Briefly, ChAd63
AMA1 was generated in suspension HEK293 cells and purified by
caesium chloride density-gradient centrifugation. MVA AMA1
was generated in chicken embryo fibroblasts (CEFs) and purified
by sucrose density-gradient centrifugation. Each vaccine lot
underwent comprehensive quality control analysis to ensure that
the purity, identity and integrity of the virus met pre-defined
specifications. Vaccine lots were stored at the clinical site at
280uC and the temperature was monitored. ChAd63 AMA1
vaccine stability was monitored by using an infectivity assay in
HEK293 cells. The immuno-potency of the MVA AMA1 vaccine
was confirmed by regular immunogenicity evaluation in mice.
Figure 1. Flow chart of the study. All vaccinations were administered intramuscularly. ChAd63 AMA1 was administered on day 0 and MVA AMA1
on day 56. Six volunteers were excluded following screening for the following reasons: psychiatric morbidity; recurrent severe urticaria; elevated
alanine aminotransferase (87 IU/L); unexplained microscopic haematuria and proteinuria; and withdrawal of consent (two individuals).
PLoS ONE | www.plosone.org3 February 2012 | Volume 7 | Issue 2 | e31208
To address the issue of AMA1 polymorphism, the transgene
insert was designed as a bi-valent composite sequence from the P.
falciparum blood-stage antigen AMA1 [19,20]. Briefly, the sequence
of the AMA1 insert contains from N- to C- terminus: the leader
sequence from human tissue plasminogen activator (tPA) followed
in-frame by the sequences encoding the ectodomain (amino acids
25–546) of P. falciparum (strain 3D7) AMA1 followed by the
ectodomain plus C-terminal transmembrane region (amino acids
25–574) of P. falciparum (strain FVO) AMA1. These two regions
represent two of the most diverged allelic variants of AMA1 (3D7
and FVO) which differ by 24 out of 622 amino acids , and
were separated in the vaccine transgene insert by a flexible linker
sequence (GGGPGGG) that has been used safely in other malaria
constructs in Phase I/IIa trials [30,32]. A number of amino acid
substitutions (9 in the 3D7 allele and 10 in the FVO allele) (Tables
S4 and S5) were also included to prevent potential N-linked
glycosylation, as described elsewhere [20,33].
Volunteers in group 1 were observed for 2 hours post each
immunization. Volunteers in group 2 were observed for 1 hour
post each immunization. Volunteers were given a digital
thermometer, injection site reaction measurement tool and
symptom diary card to record their daily temperature, injection
site reactions and solicited systemic AEs for 14 days following
vaccination with ChAd63 AMA1 and 7 days following vaccination
with MVA AMA1. Local and systemic reactogenicity was
evaluated at subsequent clinic visits and graded for severity,
outcome and association to vaccination as per the criteria outlined
in Tables S1, S2, and S3. Blood was sampled at all visits post
vaccination except days 2 and 58, and the full blood count with
differential, platelet count and serum biochemistry (including
electrolytes, urea, creatinine, bilirubin, alanine aminotransferase,
alkaline phosphatase and albumin) measured.
Peripheral Blood Mononuclear Cell (PBMC) and Serum
Blood samples were collected into lithium heparin-treated
vacutainer blood collection systems (Becton Dickinson, UK).
PBMC were isolated and used within 6 hours in fresh assays as
previously described . Excess cells were frozen in foetal calf
serum (FCS) containing 10% dimethyl sulfoxide (DMSO) and
stored in liquid nitrogen. For serum preparation, untreated blood
samples were stored at 4uC and then the clotted blood was
centrifuged for 5 min (10006g). Serum was stored at 280uC.
Peptides for T cell Assays
Peptides were purchased from NEO Peptide (Cambridge, MA,
USA). The peptides, 20 amino acids (aa) in length and overlapping
by 10 aa covered the entire AMA1 insert present in the viral
vectored vaccines. Peptides were reconstituted in 100% DMSO at
50–200 mg/mL and combined into various pools for ELISPOT
and flow cytometry assays. Additional peptide pools containing 5
peptides (tPA leader, 1 pool) or 21 peptides (‘‘Vaccine’’ and
‘‘Native’’, 1 pool for each) were prepared from pre-clinical peptide
stocks (15mers overlapping by 10 aa) as previously described .
Peptides are listed in Tables S4 and S5.
Ex-vivo interferon-c (IFN-c) ELISPOT
The kinetics and magnitude of the T cell response to AMA1
were assessed over time by ex-vivo IFN-c ELISPOT following an
18–20 hour re-stimulation of PBMC with overlapping peptides
spanning the entire AMA1 insert present in the viral vectored
vaccines (Table S4). 20mer peptides overlapping by 10 amino
acids (aa) were generated for the whole of the AMA1 vaccine insert
present in the ChAd63 and MVA vaccines. Peptides were divided
into pools containing up to 10 peptides per pool and were divided
up according to whether they were 3D7 strain specific (3 pools,
n=24), FVO specific (3 pools, n=24), common peptides (3 pools,
n=28), or FVO terminus peptides (1 pool, n=7). Fresh PBMC
were used in all ELISPOT assays using a previously described
protocol , except that 50 mL/well AMA1 peptide pools (final
concentration each peptide 5 mg/mL) were added to test wells,
50 mL/well R10 and DMSO control were added to negative un-
stimulated wells, and 50 mL/well Staphylococcal enterotoxin B
(SEB) (final concentration 0.02 mg/mL) plus phytohemagglutinin
(PHA) (final concentration 10 mg/mL) was added to positive
control wells. Spots were counted using an ELISPOT counter
(Autoimmun Diagnostika (AID), Germany). Results are expressed
as IFN-c spot-forming units (SFU) per million PBMC. Background
responses in un-stimulated control wells were almost always less
than 20 spots, and were subtracted from those measured in
peptide-stimulated wells. Responses are shown as the summed
response to all the AMA1 peptide pools (unless otherwise stated).
The tPA, ‘‘Vaccine’’ and ‘‘Native’’ responses (shown in Figure S4),
represent data measured using single pools of peptides.
Multiparameter Flow Cytometry
Cytokine secretion by PBMC was assayed by intracellular
cytokine staining (ICS) followed by flow cytometry using a
previously described protocol . Briefly, frozen PBMC were
re-stimulated for 18 hours in the presence of anti-human CD49d
and CD28 (BD Biosciences) and CD107a. Re-stimulation for the
final 16 hours was carried out in the presence of Brefeldin A
(Sigma) and Monensin (Golgi Stop, BD Biosciences). Each sample
was re-stimulated with either: 2 mg/mL SEB (positive control
samples); a pool of all 83 peptides spanning the AMA1 vaccine
antigen (see Table S4) at final concentration 2 mg/mL each
peptide and 0.11% total DMSO concentration; 0.11% DMSO
final concentration (un-stimulated
cryopreserved red blood cells infected with schizont/late tropho-
zoite stage 3D7 strain P. falciparum parasites (iRBC) at 56106/mL;
or uninfected red blood cell controls (uRBC) at 56106/mL
prepared in the same manner. Cells were stained the next day
using a Live/Dead marker, as well as for CD4, CD14, CD20,
CD8a, CD3, IFN-c, TNFa, and IL-2. Samples were analyzed
using a LSRII Flow Cytometer (BD Biosciences) and FlowJo v8.8
(Tree Star Inc, USA). Dead cells, monocytes (CD14+), and B cells
(CD20+) were excluded from the analysis. The Boolean gate
platform was used with individual gates to create response
combinations (Figure S6). Analysis and presentation of distribu-
tions was performed using SPICE v5.2, downloaded from http://
exon.niaid.nih.gov/spice . Background responses in un-
stimulated peptide and uRBC control cells were subtracted from
the AMA1 peptide and iRBC stimulated responses respectively.
peptide control sample);
Total IgG ELISA
The recombinant 3D7 AMA1 protein was a gift from Dr
Chetan Chitnis (ICGEB, New Delhi, India) and FVO AMA1 was
a gift from Dr Mike Blackman (NIMR, London, UK) .
ELISAs were performed with these proteins using the same
standardized methodology, as previously described for the MSP119
antigen . The reference serum used to generate the standard
curve was prepared from adult Kenyan immune serum (a gift from
Dr Faith H. Osier, KEMRI-Wellcome, Kilifi, Kenya). Antibodies
against AMA1 (3D7 and FVO alleles) were also assayed by the
GIA Reference Center (NIH, USA) as previously described ,
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and these OD-based ELISA units were converted to antigen-
specific mg/mL also as previously described .
In vitro Assay of Growth Inhibitory Activity (GIA)
The ability of induced anti-AMA1 antibodies to inhibit growth
of P. falciparum 3D7 and FVO strain parasites was assessed by a
standardized GIA assay using purified IgG as previously described
. Briefly, each test IgG (10 mg/mL in a final test well) was
incubated with synchronized P. falciparum parasites for a single
growth cycle and relative parasitemia levels were quantified by
biochemical determination of parasite lactate dehydrogenase.
Measurement of NAb Titers to ChAd63
ChAd63 antibody neutralization assays were performed as
previously described  except that a ChAd63 vector expressing
secreted alkaline phosphatise (SEAP) , and GripTiteTM 293
MSR cells (Invitrogen R795-07) were used and cultured for one
day prior to infection. The lowest serum dilution tested was 1:18
and thus samples that did not give 50% neutralization in
comparison to the control at this level are reported as ‘‘negative’’
Data were analyzed using GraphPad Prism version 5.03 for
Windows (GraphPad Software Inc., California, USA). Geometric
mean or median responses for each group are described.
Significance testing of differences between two groups used the
two-tailed Mann-Whitney U test or Wilcoxon signed rank test as
appropriate. Correlations were analyzed using Spearman’s rank
correlation co-efficient (rs) for non-parametric data. A value of
P,0.05 was considered significant.
Recruitment took place between February 2010 and June 2010.
Sixteen healthy malaria-naı ¨ve adult volunteers (10 female and 6
male) were enrolled, immunized and followed up (Figure 1). The
mean age of volunteers was 30 years (range 18–48). Vaccinations
began in March 2010 and all follow-up visits were completed by
October 2010. All volunteers attended all visits as scheduled and
completed the study.
Safety and Reactogenicity
No unexpected or serious AEs occurred and no volunteers were
withdrawn due to AEs. ChAd63 AMA1 demonstrated a good
safety profile with the majority of AEs mild in severity (89%), all
resolving completely, most (63%) within 48 hours (Figure 2).
Overall, 12 out of 16 volunteers (75%) experienced one or more
local AEs related to ChAd63 AMA1; these were mild with the
exception of three cases of arm pain (two moderate and one
severe), one case of moderate swelling and one case of moderate
erythema, all occurring in the higher dose group (Figure 2A). 10
out of 16 volunteers (63%) also experienced at least one or more
systemic AEs related to vaccination. These were all mild with the
exception of two cases of severe malaise and one case of severe
headache experienced by two volunteers in the higher dose group
(Figure 2B). MVA AMA1 administered intramuscularly at the
relatively high poxviral dose of 56108pfu to the first four boosted
volunteers (group 1B), was markedly more reactogenic than
ChAd63 AMA1, with 3 out of 4 vaccinees (75%) experiencing a
constellation of severe ‘flu-like’ systemic AEs (including malaise,
myalgia, rigor, fatigue and feverishness) (Figure 3A). One of these
volunteers described disorientation and ‘visual hallucinations’
whilst experiencing feverishness and a recorded fever of 37.8uC
(AE defined as ‘delirium’). 92% of severe symptoms resolved
completely within 48 hours of onset (one case of severe arthralgia
took 72 hours to resolve). MVA expressing other malaria or HIV
antigens has been used previously at doses equal to and higher
than 56108pfu without safety concern [39,40], and such a dose
was used because antibody induction in pre-clinical models has
been shown to be dose dependent [41,42]. However, after
consultation with the Local Safety Committee, the dose of MVA
AMA1 was subsequently halved to 2.56108pfu for the fifth
boosted volunteer but still resulted in severe local and systemic AEs
(malaise and headache resulting in absenteeism from work) (group
2B (i), Figure 3B). The dose was therefore further reduced by half
again to 1.256108pfu for the remaining three volunteers (group
2B (ii), Figure 3C) and this now demonstrated an acceptable
reactogenicity profile, typical of the MVA vector [39,43], inducing
only mild systemic AEs and mild or moderate local AEs.
ChAd63-MVA AMA1 T cell immunogenicity assessed by
ex-vivo IFN-c ELISPOT
Vaccination with ChAd63-MVA AMA1 induced antigen-
specific T cell responses in all volunteers as measured by ex-vivo
IFN-c ELISPOT using 20mer peptides overlapping by 10aa, with
individual responses shown in Figure S1 and median responses to
the total vaccine insert shown for each group in Figure 4A and B.
Following ChAd63 AMA1 prime, there was no significant
difference between median responses in the higher dose group 2
in comparison to group 1 at the peak of the response on day 14
(median 921 [range 318–1366] vs 933 [range 298–2942] SFU/
million PBMCs in groups 2 versus 1 respectively, n=8 vs 8,
P=0.79 by Mann-Whitney test). Responses subsequently followed
a classical T cell kinetic and contracted by day 56 (Figure 4A).
Administration of MVA AMA1 at day 56 significantly boosted
these responses in all volunteers as measured one week later on
day 63 (Figure 4B). Due to the differences in dosage of MVA at
this time point it is difficult to directly compare groups, however
the volunteers in group 1B who all received 56108pfu MVA
AMA1 demonstrated a higher median response than those
receiving the lower doses of MVA AMA1 (7186 [range 4372–
10832] vs 2631 [range 2102–4500] SFU/million PBMCs). This
was primarily due to two very strong responders in group 1B with
peak post-boost responses in the region of 10,000 SFU/million
PBMC. Analysis of the breakdown of the day 63 ELISPOT data
showed that T cells were induced to both alleles equally; 3D7
(median 4959 [group 1B], 2036 [group 2B] SFU/million PBMC)
and FVO (median 4359 [group 1B], 1539 [group 2B] SFU/
million PBMC) (Figure 4C). Responses again contracted but were
maintained above baseline at the end of the study period (day 140)
with similar overall result to that observed at day 63 (Figure 4D).
Breadth of the AMA1 T cell response
T cell responses in all volunteers were detected in multiple
peptide pools spanning the entire AMA1 vaccine insert in the ex-vivo
IFN-c ELISPOT assay. Individual responses are shown according
to magnitude of the response (Figure S2) or as a percentage of the
total summed ELISPOT response (Figure S3). Irrespective of
whether the responses are analyzed after the priming immunization
with ChAd63 AMA1 or following the MVA AMA1 boosting
immunization, the individual and median responses broadly mirror
the composition of the vaccine antigen (Figure S2A). These data
indicate that no single immuno-dominant region exists within the
AMA1 transgene insert and importantly that responses were
induced to epitopes contained within peptides whose sequence is
common to both alleles. T cell responses were also measured to the
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tPA leader sequence (Figure S4A), and this showed the absence of
any response induction against this mammalian sequence. A total of
19 amino acid substitutions were also included in the AMA1
transgene to remove sites of potential N-linked glycosylation. A pool
of 21 previously described peptides (15mers overlapping by 10aa,
Table S5) was used that corresponds only to those peptides that
peptides represent 21 out of 170 peptides (15mers) that are required
to cover the whole AMA1 transgene insert, i.e. 12%. These two
single pools were used here to measure responses following human
sequences of AMA1 showed a significantly stronger response to the
vaccine pool for both day 14 (n=16, P=0.0007) and day 63 (n=8,
P=0.008) as assessed by Wilcoxon signed rank test (Figure S4B).
AMA1 T cell multi-functionality
Antigen-specific CD3+T cell functionality was also assayed by
ICS at the day 84 time-point (Figure 4E,F). Following peptide re-
stimulation, detectable AMA1-specific CD3+T cells consisted of a
mixed CD4+and CD8+phenotype. It should be noted that the
ELISPOTand ICSassays vary inmethodology(includingthe use of
multiple versus a single peptide pool respectively, as well as
differences in peptide concentration, use of co-stimulatory antibod-
ies and use of fresh versus frozen PBMC). Nevertheless, in
agreement with the ex-vivo IFN-c ELIspot data for this time-point
(Figure 4A,B), reasonably comparable responses were seen in
groups 1B and 2B for both T cell subsets. CD8+T cells upregulated
CD107a expression (marker of degranulation), and produced IFN-c
and TNFa but only negligible levels of IL-2. In comparison the
CD4+T cells produced high levels of TNFa, lower levels of IFN-c
and IL-2, but did not upregulate CD107a expression. However,
following re-stimulation with cryopreserved red blood cells infected
with schizont/late trophozoite stage 3D7 strain P. falciparum
parasites (iRBCs), negligible (,0.05%) CD4+or CD8+T cell
responses were evident above background uRBC re-stimulation.
This is in contrast to results seen following ChAd63-MVA MSP1
Figure 2. Local and systemic AEs deemed definitely, probably or possibly related to ChAd63 AMA1. Only the highest intensity of each
AE per subject is listed. Data are combined for all AEs for all volunteers receiving the same vaccine at the stated dose. There were no immunization
related serious AEs. (A) Local AEs post ChAd63 AMA1. (B) Systemic AEs post ChAd63 AMA1. ‘Other’ AEs post 56109vp ChAd63 AMA1 included
cough, coryzal symptoms, abdominal pain and dysmenorrhoea. ‘Other’ AE post 561010vp ChAd63 AMA1 was coryzal symptoms. All ‘other’ AEs were
considered possibly related to vaccination due to a temporal association.
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Figure 3. Local and systemic AEs deemed definitely, probably or possibly related to MVA AMA1. Only the highest intensity of each AE
per subject is listed. Data are combined for all AEs for all volunteers receiving the same vaccine at the stated dose. There were no immunization
related serious AEs. (A) Local and systemic AEs post 56108pfu MVA AMA1. Local ‘other’ AE was mild bruising at vaccination site. Systemic ‘other’ AEs
included two cases of rigor, one case of delirium, loss of appetite and chills. (B) Local and systemic AEs post 2.56108pfu MVA AMA1. ‘Other’ AE post
2.56108pfu MVA AMA1 was dizziness. (C) Local and systemic AEs post 1.256108pfu MVA AMA1.
PLoS ONE | www.plosone.org7 February 2012 | Volume 7 | Issue 2 | e31208
Figure 4. Cellular immunogenicity of ChAd63 AMA1 and ChAd63-MVA AMA1 immunization regimes. (A) Groups 1A and 2A and (B)
groups 1B and 2B median ex-vivo IFN-c ELISPOT responses in PBMC to the AMA1 insert (summed response across all the individual peptide pools).
Individual responses are shown in Figure S1. Note due to allele-specific peptide overlap some responses can be potentially counted twice. Individual
breakdowns of the ELISpot responses are shown in Figure S2, and data in Figure S3C show that on average one quarter to one third of the total
summed response can be attributed to FVO allele-specific peptides. (C) Median and individual IFN-c ELISPOT responses at day 63 and (D) at day 140
that are functional to the individual alleles 3D7 (circles) and FVO (squares). (E,F) PBMC from day 84 for group 1B (closed symbols) and group 2B (open
symbols) were re-stimulated with a pool of AMA1 peptides or cryopreserved iRBCs. Individual data points and the median are shown for (E) the %
CD4+and (F) CD8+T cells positive for CD107a, IFN-c, IL-2 or TNFa. The dotted line indicates the 0.05% level and any response ,0.03% is not shown.
PLoS ONE | www.plosone.org8 February 2012 | Volume 7 | Issue 2 | e31208
immunization , whereby iRBC re-stimulation of PBMC
resulted in comparable detection of CD4+(but not CD8+) MSP1-
specific T cell responses to those seen following peptide re-
stimulation. This result may reflect the lower levels of AMA1
antigen present in iRBC preparations that is available for
presentation to T cells (in comparison to the more abundant
merozoite surface protein), or may in part be due to the
aforementioned aminoacid substitutionsaffectingT cellrecognition
of native parasite AMA1 antigen. Distinct populations of CD4+and
CD8+T cells expressing 1+, 2+, 3+ or 4+ functional markers/
cytokines wereevident followinga Boolean gate analysis (FigureS5).
ChAd63-MVA AMA1 antibody immunogenicity assessed
The kinetics and magnitude of the serum IgG antibody response
against the 3D7 allele of AMA1 were assessed over time by
ELISA. AMA1-specific IgG was induced in all volunteers, with
individual responses shown in Figure S7 and geometric mean
(geomean) responses for each group in Figure 5A,B. Following the
ChAd63 AMA1 prime, there were significantly stronger responses
in the higher dose group 2 in comparison to group 1 at the peak of
the response on day 28 (geomean titer 109 [range 48–196] vs 37
[range 18–192] AMA1 antibody units (AU) in groups 2 versus 1
respectively, n=8 vs 8, P=0.01 by Mann-Whitney test).
Responses declined slowly but were maintained above the
detection limit at day 90 in groups 1A and 2A. Administration
of MVA AMA1 at day 56 significantly boosted these responses in
all volunteers bar one (in group 2B(ii)), with serum IgG responses
peaking four weeks later as measured on day 84. At this time-
point, there was no significant difference (P=0.68 by Mann-
Whitney test) in the average AMA1-specific IgG responses in
group 2B (561010vp ChAd63 AMA1 prime and 2.5 or
1.256108pfu MVA AMA1) in comparison to group 1B (56109
vp ChAd63 AMA1 prime and 56108pfu MVA AMA1) (geomean
titer 1709 [range 840–3370] vs 949 [range 84–2552] AMA1 AU
respectively). Responses again declined over time but were
maintained at high levels at the end of the study period (day
140) with again no significant difference (P=1.00 by Mann-
Whitney test) in the responses between groups 1B and 2B
(geomean titer 971 [range 553–2683] vs 547 [range 90–1162]
AMA1 AU respectively, n=4 vs 4). Serum IgG responses against
the FVO allele of AMA1 (which differs from the 3D7 allele by 24
amino acids) were also assessed by ELISA at day 84, and a strong
correlation was evident between the responses against the two
alleles (Spearman r=0.97, P=0.0004) (Figure S8A).
AMA1 AU quantified by standardized ELISA were also
converted to concentration of antigen-specific antibodies against
both alleles of AMA1 , with median concentrations of 58 mg/
mL and 62 mg/mL anti-AMA1 3D7 and FVO IgG, respectively,
in group 1B and 49 mg/mL and 41 mg/mL anti-AMA1 3D7 and
FVO IgG, respectively, in group 2B (Figure 5C). The AMA1
specific IgG response induced functional GIA above baseline
against both the 3D7 and FVO strains of P. falciparum in vitro
(Figure 5D) and there was a significant correlation between the
3D7 and FVO % GIA (Spearman r=0.90, P=0.005) (Figure
S8B). A sigmoidal relationship between 3D7 AMA1 AU and %
3D7 parasite GIA was also evident (Figure 5E), as described in
other studies [20,36].
Pre-existing NAb titers against ChAd63 and
NAb titers against ChAd63 were assayed in the pre-immuni-
zation (day 0) serum of all volunteers but these were not used as an
exclusion criterion. Five out of 16 volunteers were negative
(titer,1:18) and of the remaining 11 individuals all were low with
the exception of two who had titers.1:200 (the highest being
1:369). There was no significant difference between the volunteers
enrolled into Group 1 or Group 2 (n=8 per group, P=0.18,
Mann Whitney test) (Figure S9A). There were also no significant
correlations between these low level pre-existing NAb responses
and ChAd63 AMA1 immunogenicity (as assessed by ELISPOT or
ELISA at the peak of the priming response) in either dose group
In this Phase Ia study we have shown in healthy, malaria-naı ¨ve
adult volunteers that a recombinant ChAd63-MVA heterologous
prime-boost immunization regimen encoding AMA1 can induce
functional antigen-specific antibody responses in addition to strong
T cell responses. ChAd63 AMA1 demonstrated a good reactoge-
nicity profile, similar to that seen consistently with the same doses
of ChAd63 vectored vaccines encoding the pre-erythrocytic
malaria antigen ME-TRAP (O’Hara et al., J Infect Dis 2011 in
press) and the blood-stage antigen MSP1 , supporting the
growing body of evidence that this simian adenovirus vector is safe
for clinical use. MVA expressing other malaria or HIV antigens
has been used previously as a vaccine vector  at doses equal to
and higher than the maximum dose used in this study without
safety concern [39,40]. The increased reactogenicity seen with the
relatively high poxviral doses of MVA AMA1 used in this study
(56108pfu and 2.56108pfu) may be a result of differences in viral
titration methods or other factors. When MVA AMA1 was used at
a dose of 1.256108pfu, an acceptable reactogenicity profile was
observed, without a significant compromise to vaccine antibody
immunogenicity, although T cell responses tended to be higher
following the higher boosting dose of MVA. A subsequent study
has now shown this acceptable tolerability profile to be maintained
following immunization of a further nine volunteers with the
dosing regimen used here in group 2B(ii) (Sheehy et al., manuscript
The summed AMA1-specific IFN-c T cell responses peaked at
a median level of .6900 SFU/million PBMC in group 1B
(ChAd63 56109vp and MVA 56108pfu), .2500 SFU/million
in group 2B (ChAd63 561010vp & MVA 1.25–2.56108pfu).
However, it remains possible that some T cell epitopes are
duplicated within the 3D7- and FVO-specific 20mer peptides
(because a number of these only differ by 1–2 amino acids) and
thus the total sum may potentially count some responses twice.
Examining the median summed individual responses to the two
alleles in the vaccine, we found T cells were induced to both
alleles equally; 3D7 (4959 SFU/million PBMC in group 1B and
2036 SFU/million PBMC in group 2B) and FVO (4359 SFU/
million PBMC in group 1B and 1539 SFU/million PBMC in
group 2B). The same result was observed at the late day 140
time-point. These allele-specific totals are not subject to any
double counting error due to peptide homology between the 3D7
and FVO strains, and these median ELISPOT responses
compare favourably with those induced by the same vectors
and regimen for the ME-TRAP and MSP1 malaria antigens and
are substantially greater than those reported for AMA1 protein-
in-adjuvant vaccines [44,45]. Responses induced after the
ChAd63 AMA1 immunization also compared favourably with
those recently reported following a single immunization of
healthy adult US volunteers with 161010vp of an AdHu5
vaccine encoding the 3D7 AMA1 antigen , although
interestingly in this study the use of a higher dose (561010vp)
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led to a significant reduction in T cell immunogenicity – an
observation not seen here with the same dose of ChAd63.
A total of 19 amino acid substitutions, as described and used
elsewhere [20,33], were also included in the vaccine transgene to
remove sites of potential N-linked glycosylation that could occur
during in vivo mammalian expression of the AMA1 transgene.
Such amino acid substitutions could affect T cell recognition of
the native parasite AMA1 sequence if they occurred within
recognized epitopes. Following immunization of rhesus ma-
caques with the same ChAd63-MVA AMA1 vaccines these
Figure 5. Antibody immunogenicity of ChAd63 AMA1 and ChAd63-MVA AMA1 immunization regimes. (A) Groups 1A and 2A and (B)
groups 1B and 2B total IgG ELISA responses against 3D7 AMA1 as measured in the serum over time. The geometric mean response is shown for each
group and individual responses are shown in Figure S7. The horizontal dotted line indicates the limit of detection of the assay. (C) AMA1-specific
ELISA titers against 3D7 (circles) and FVO (squares) in mg/mL for group 1B (n=4) and group 2B (n=4) at the peak time-point (day 84). Individual data
points and the median are shown. (D) % GIA against 3D7 and FVO parasites at day 0 and day 84 in group 1B (open symbols) and 2B (closed symbols).
(E) Relationship between 3D7 strain % GIA and serum 3D7 AMA1-specific IgG ELISA titer at day 0 and day 84 in groups 1B (open symbols) and 2B
PLoS ONE | www.plosone.org10 February 2012 | Volume 7 | Issue 2 | e31208
substitutions were shown to have a minimal effect following
comparative re-stimulation with peptides representing these
substitutions and which reflected about 12% of the total number
of 15mer peptides required for complete coverage of the AMA1
transgene . However following human vaccination here,
responses to peptide pools for the vaccine versus native
sequences of AMA1 showed a significantly stronger response to
the vaccine pool, indicating amino acid substitutions included in
the insert are likely to impact on the T cell recognition of native
AMA1 parasite antigen in humans – an effect potentially
observed in the peptide versus pRBC re-stimulation of PBMC.
Similarly, it cannot be automatically assumed that cellular
immune responses to the native AMA1 sequence would have
been as strong as those observed to the modified vaccine
sequence, although given responses were broadly strong to the
remaining native sequence of the antigen (roughly 88% of the
total), this seems unlikely. Future studies should thus extremely
carefully address the potential advantages and disadvantages of
such modifications in the context of vectored vaccine design.
The discrepancy described here between the T cell data in
rhesus macaques versus humans, indicates that the assessment of
the positive versus negative impact(s) of such modifications at the
preclinical stage may be extremely difficult.
The CD3+T cell populations also consisted of a mixed CD4+
and CD8+phenotype, and the cells expressed a range of functional
cytokines/markers following AMA1 peptide re-stimulation in vitro.
It remains a significant challenge to the field to establish the
relevance of such in vitro measurements of T cell phenotypes to
protective outcome against malaria in vivo in humans. Similar
challenges are faced for in vitro assays of antibody function, and
ultimately the development of an effective vaccine is required
before such questions can be readily addressed. Nevertheless, the
ChAd63-MVA vaccine platform, now tested here for the third
time, represents a versatile delivery system for the reliable
induction of high-level antigen-specific multi-functional T cell
responses. The adverse reactogenicity profile of higher doses of
MVA AMA1 means that future trials will use a maximum dose of
1.256108pfu of this vaccine, which will limit median T cell
responses to approximately 2000 SFU/million PBMC. However,
these data indicate that exceptionally strong T cell responses
(approximately 10,000 SFU/million PBMC) have the potential to
be induced in humans by subunit vaccination, if the MVA vector
could be further engineered or formulated to reduce reactogenicity
whilst maintaining transgene potency.
In agreement with pre-clinical data in mice, rabbits and rhesus
macaques [19,20], this ChAd63-MVA prime-boost regimen also
induced substantial AMA1-specific serum IgG antibody responses.
When quantified by a standardized ELISA, the concentrations of
AMA1-specific IgG induced at the peak of the response (median
49 mg/mL against 3D7 AMA1 in group 2B) were comparable to
AMA1 protein vaccines formulated in Alum  or Montanide
ISA720 , but three- to four-fold lower than Alum+CpG
[45,48]. As for T cell induction, the magnitude of these antigen-
specific IgG antibody responses is also highly comparable to those
seen with the ChAd63-MVA MSP1 regime . Importantly,
however, unlike for MSP1, ChAd63-MVA AMA1 induced
moderate growth inhibition in vitro that correlated with total IgG
ELISA titers. These levels were higher than those seen with a bi-
valent (3D7 and FVO) AMA1 protein vaccine (AMA1-C1)
formulated in Alum  and an AdHu5 vaccine encoding the
3D7 allele of AMA1  (and assessed using the same assay of
GIA), comparable to those when the vaccine was formulated in
Montanide ISA720 , but lower than those seen with the same
vaccine was formulated in Alum+CpG [45,48]. This ability of the
AMA1 antigen to induce functional GIA at lower levels of antigen-
specific human IgG (in comparison to MSP1) is in agreement with
previous data generated with protein-in-adjuvant vaccines .
Similarly, in this study in vitro inhibition levels of the FVO strain
parasite were lower than those observed for 3D7, which is in
agreement with previous clinical studies of the AMA1-C1 protein-
in-adjuvant vaccine [47,48,49]. Interestingly, dose escalation of
ChAd63 AMA1 from 56109vp to 561010vp led to a statistically
significant increase in AMA1-specific serum IgG antibody
responses but no significant increase in AMA1-specific IFN-c T
cell responses. The same finding was also seen with ChAd63
MSP1 . A recent report of an AdHu5 vectored malaria
vaccine showed the same trend for antibody responses but
significantly reduced T cell responses at the highest dose used
, whereas dose finding studies with the MRKAd5-gag HIV
vaccine showed a similar plateauing of T cell responses (as assessed
by ex-vivo IFN-c ELISPOT) at the highest doses used . The
reason(s) for these quantitative T cell differences at high vaccine
dose remain unclear and such responses may be vector as well as
antigen-dependent. Similarly, functional or qualitative differences
in T cell effector function following adenoviral vaccine dosing
warrants further investigation.
In agreement with data relating to the use of the ChAd63 ME-
TRAP vaccine in healthy UK volunteers (O’Hara et al., J Infect
Dis 2011 in press), there was also no apparent effect of low-level
pre-existing anti-ChAd63 NAb responses on AMA1-specific
cellular or humoral immune responses. This is also perhaps
unsurprising given the relatively high doses of adenoviral vaccine
used. Similar findings have recently been reported in healthy US
adults following a single immunization with an AdHu5 vaccine
encoding the P. falciparum circumsporozoite antigen .
The T cell and GIA data are reassuringly comparable to those
seen in pre-clinical studies in mice [19,42,52] and macaques ,
supporting the importance of these models in pre-clinical vaccine
optimization and development. The data also confirm that in
comparison with leading AMA1 protein-in-adjuvant vaccines, the
ChAd63-MVA vectored vaccine regimen in humans can induce
comparable antibody titers, similar degrees of in vitro growth
inhibition and markedly stronger CD4+and CD8+T cell
responses including those against conserved sequences of AMA1.
Since AMA1 is also expressed on sporozoites and during the liver-
stage of infection [21,53], the strong T cell responses, in particular
CD8+T cells, induced by ChAd63-MVA AMA1 may reduce the
parasite inoculum released from the liver  and thus increase
the potential for clinical efficacy at the blood-stage. It has been
shown in animal models that immunization with multiple AMA1
alleles can focus B cell responses on more conserved eptiopes ,
and similarly the inclusion of conserved T cell epitopes in the
vaccine insert described here (if capable of contributing to
protective immunity in humans) may help to address the induction
of effective immunity in the face of extensive parasite genetic
variability. However, it remains for now unclear how effective
these vaccines would be in the context of the marked AMA1
polymorphisms that are commonly seen in infecting parasites in
the field  that are highly likely to affect the neutralizing ability
of vaccine-induced antibodies. Future work should also examine
whether the enhanced cell-mediated and humoral immunogenicity
reported in pre-clinical studies (when protein-in-adjuvant formu-
lations are combined with viral vectored vaccines [20,41,55]) can
also be achieved in humans in clinical trials. Further studies are
now essential to ascertain whether the addition of strong cellular
immunity to such levels of antibody response can translate into
significant vaccine efficacy against blood-stage infection in Phase
IIa controlled human malaria challenge trials.
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SPOT responses to the AMA1 insert (summed response across all
the individual peptide pools) are shown over time following
immunization in (A) Group 1A (n=4), (B) Group 1B (n=4), (C)
Group 2A (n=4), and (D) Group 2B (n=4). Individual responses
are shown for each volunteer. MVA dosage explained in Figure 1.
In Group 2B volunteer I=2B (i), volunteers II–IV=2B (ii). Note
due to allele-specific peptide overlap some responses can be
potentially counted twice. Individual breakdowns of the ELISpot
responses are shown in Figure S2, and data in Figure S3C show that
on average one quarter to one third of the total summed response
can be attributed to FVO allele-specific peptides.
Individual ex-vivo IFN-c ELISPOT data. ELI-
according to total response. (A) The % of the amino acid
sequence within the AMA1 vaccine insert that is attributable to
each peptide pool is shown. (B) Data show the total response to
each peptide pool within the AMA1 insert for each volunteer at
day 14 after ChAd63 AMA1 immunization. (C) Data show the
total response to each peptide pool within the AMA1 insert for
each volunteer at the peak of the response at day 63 after ChAd63-
MVA immunization. (D) The median IFN-c response to each
peptide pool within the AMA1 insert according to group (G1 or
G2) and immunization regime (Ad=ChAd63, AdM=ChAd63-
MVA) at the peak time-point (day 14 after ChAd63 prime and day
63 after ChAd63-MVA prime-boost). MVA dosage explained in
Figure 1. Volunteer I=2B (i), Volunteers II–IV=2B (ii).
Breakdown of ex-vivo IFN-c ELISPOT data
according to % response. (A) Data show the % of the total
response to each peptide pool within the AMA1 insert for each
volunteer at day 14 after ChAd63 immunization. (B) Data show
the % of the total response to each peptide pool within the AMA1
insert for each volunteer at the peak of the response (day 63) after
ChAd63-MVA immunization. (C) The median response to
peptide pool within the AMA1 insert according to group (G1 or
G2) and immunization regime (Ad=ChAd63, AdM=ChAd63-
MVA) at the peak time-point (day 14 after ChAd63 prime and day
63 after ChAd63-MVA prime-boost). MVA dosage explained in
Figure 1. Volunteer I=2B (i), Volunteers II–IV=2B (ii).
Breakdown of ex-vivo IFN-c ELISPOT data
Native pools. (A) Data show ex-vivo IFN-c ELISPOT responses
to the tPA peptide pool for all volunteers at each time-point during
the trial (n=16 for d0–d56; n=8 for d63–d140). Individual data
points are shown as well as the median. Nominal cut-off for a
significant response marked as 50 SFU/million PBMC. (B) Data
show ex-vivo IFN-c ELISPOT responses to peptide pools for
Vaccine and Native sequences for all volunteers at the peak time-
point after the prime (n=16) and peak time-point after the boost
(n=8). Individual data points and median are shown. The
responses shown represent data measured using a single pool of
peptides (n=21 peptides in each). In each graph: Group 1A (open
circles); Group 1B (open squares); Group 2A (closed circles);
Group 2B (closed squares).
T cell responses to tPA sequence, Vaccine and
MVA AMA1 immunization. The multi-functionality of the
T cell multi-functionality following ChAd63-
CD3+T cell responses was assessed by polychromatic flow
cytometry and ICS. Frozen PBMCs from day 84 were re-
stimulated with a pool of AMA1 peptides and cells were stained as
described. Gating strategy and representative plots are shown in
Figure S6. The multi-functional compositions of the T cell
responses following ChAd63-MVA immunization are shown for
(A) CD4+and CD8+T cells in Group 1B following AMA1 peptide
re-stimulation, and (B) CD4+and CD8+T cells in Group 2B
following AMA1 peptide re-stimulation. Responses are grouped
and colour-coded according to the CD4+and CD8+subsets, and
the number of functions detected for each T cell population.
Individual data points and median percentage of the parent CD4+
or CD8+response (open bars) are shown for each of the functional
populations indicated on the x-axis. The pie charts summarize the
fractions of AMA1-specific CD4+or CD8+T cells that are positive
for a given number of functions.
T cell responses. Representative flow cytometry plots are
shown for the analysis of AMA1-specific T cell responses from
volunteers immunized with ChAd63-MVA AMA1. (A) Initial
gating used (from top left to bottom right) forward scatter area
(FSC-A) versus forward scatter height (FSC-H) to remove doublet
events and select singlet cells; then following this small lympho-
cytes were gated using FSC-A versus side scatter area (SSC-A);
then live CD142CD202CD3+cells were selected; then CD4
versus CD8 was used to select the total CD4+CD82cell
population and vice versa for the CD8+CD42population.
Cytokine (IFN-c, IL-2 and TNFa) and CD107a gating using
bivariate plots is shown for (B) CD4+cells and (C) CD8+cells. (B)
Representative plots for un-stimulated (UNS), AMA1 peptide
stimulated (AMA1), SEB, uRBC and iRBC stimulated samples are
shown. IFN-c (top row), IL-2 (second row), TNFa (third row) and
CD107a (bottom row) for the CD82CD4+T cell population were
analyzed using bivariate plots. Percentages refer to the % of CD82
cells that express the specific cytokine or marker.
Background responses in UNS or uRBC control cells were
subtracted from the AMA1 and iRBC response respectively during
the analysis. (C) Same analysis as in (B), except for the CD42
CD8+T cell population.
Gating strategy for analysis of AMA1-specific
responses against 3D7 PfAMA1 as measured in the serum over
time following immunization in (A) Group 1A (n=4), (B) Group
1B (n=4), (C) Group 2A (n=4), and (D) Group 2B (n=4). MVA
dosage explained in Figure 1. Volunteer I=2B (i), Volunteers II–
Individual IgG ELISA data. Total IgG ELISA
and % GIA. (A) Spearman’s correlation of serum IgG ELISA
titers against AMA1 for the 3D7 versus FVO alleles at day 84,
n=8. (B) Relationship between 3D7 strain % GIA and FVO strain
% GIA using purified IgG at 10 mg/mL.
Relationship between total IgG ELISA titers
titers against ChAd63 were assayed in the pre-immunization (day
0) serum of all volunteers. (A) Individual and median responses in
Groups 1 and 2 are shown. The dotted lines indicate the level at
which responses were classed as negative (titer,1:18) and high
(titers.1:200). There was no significant difference between the
volunteers enrolled into Group 1 or Group 2 (n=8 per group,
P=0.18, Mann Whitney test). There were no significant
Baseline NAb responses against ChAd63. NAb
PLoS ONE | www.plosone.org 12February 2012 | Volume 7 | Issue 2 | e31208
correlations in either (B) Group 1 or (C) Group 2 between these
low level pre-existing NAb titers and ChAd63 AMA1 immuno-
genicity as assessed by peak total summed AMA1 ELISPOT
response at day 14 (blue) or anti-AMA1 (3D7) total IgG ELISA at
day 28 (red). These were selected as the peak of the priming
immune responses following ChAd63 AMA1 immunization.
rs=0.36, P=0.39 (ELISA) or rs=20.53, P=0.20 (ELISPOT) in
Group 1; and rs=0.32, P=0.43 (ELISA) or rs=20.02, P=0.98
(ELISPOT) in Group 2.
Supplementary Information S1
criteria for volunteers in study.
Inclusion and exclusion
Assessment of severity of AEs.
Assessment of severity of local AEs.
Assessment of relationship of AE to vaccina-
overlapping by 10 amino acids (aa) were generated for the whole
of the AMA1 vaccine insert present in the ChAd63 and MVA
vaccines. Peptides were divided into pools containing up to 10
peptides and were divided up according to whether they were 3D7
strain specific (3 pools, n=24), FVO specific (3 pools, n=24),
AMA1 overlapping peptides. 20mer peptides
common peptides (CP; 3 pools, n=28), FVO terminus peptides
(FVOT; 1 pool, n=7). A single pool of tPA peptides (n=5) was
used and these were 15mers overlapping by 10aa. Amino acids
that were substituted to prevent potential N-linked glycosylation
are indicated in bold.
sequences are shown, and pools indicated: V=sequence found in
vaccine insert (n=21); N=sequence found in native parasite
(n=21). Amino acids that were substituted to prevent potential N-
linked glycosylation are highlighted in bold.
Vaccine versus Native peptides. 15mer peptide
We thank C. Bateman, M. Smith, R. Singzon and J. Ryu for clinical
assistance; L. Dinsmore for logistical support; M. Cottingham, S. Saurya, J.
Furze, C. Oliveira, E. Bolam, E. Mukhopadhyay, A. Crook, A. Turner and
N. Green for assistance with vaccine manufacture; the Jenner Institute
Flow Cytometry and Vector Core Facilities for technical assistance; S.
Moretz, H. Zhou and G. Tullo for technical support performing the GIA
assays; and all the study volunteers.
Conceived and designed the experiments: GAO AML AVSH SJD.
Performed the experiments: SHS CJAD SCE SB KAC FDH KJE TM AJS
IDP MDJD NE KG CAL AML TD SJD KM. Analyzed the data: SHS
SCE SB SJD. Contributed reagents/materials/analysis tools: EB SM SC
RC SCG AN. Wrote the paper: SHS SCE SB AVSH SJD. Project
Management: AML KG.
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